Device and method for measuring impedance in organotypic tissues

ABSTRACT

The present invention refers to a device for measuring impedance in organotypic tissue comprising at least one recording chamber with a liquid permeable membrane supporting the organotypic tissue, at least one bottom electrode and at least one top electrode, wherein the liquid permeable membrane divides the recording chamber into a top chamber and a bottom chamber, wherein at least the bottom chamber contains culture medium for the organotypic tissue, and the bottom electrode(s) is/are located in the bottom chamber and the top electrode(s) is/are located in the top chamber, and wherein the organotypic tissue is located between the bottom electrode(s) and the top electrode(s). The present invention also refers to the use of the device according to the present invention for measuring impedance in organotypic tissue. 
     Furthermore, the present invention relates to a method for analyzing the effect of test compounds on pathological and non-pathological organotypic tissue by measuring the impedance of the organotypic tissue, wherein the organotypic tissue is cultured in a culture medium during the time of the analysis and the impedance of the organotypic tissue is measured at least once before and at least once after treating the organotypic tissue with the test compound or the impedance of the organotypic tissue treated with the test compound is compared to a non-treated organotypic tissue, wherein the impedance is measured using at least one electrode at each of two opposing sides of the organotypic tissue, and the electrodes are contacted with the culture medium or the tissue during measuring the impedance.

This application claims priority to European Patent Application No.08.003365.7 filed Feb. 25, 2008, the contents of which are incorporatedby reference herein in its entirety.

The present invention refers to a device for measuring impedance inorganotypic tissue comprising at least one recording chamber with aliquid permeable membrane supporting the organotypic tissue, at leastone bottom electrode and at least one top electrode, wherein the liquidpermeable membrane divides the recording chamber into a top chamber anda bottom chamber, wherein at least the bottom chamber contains culturemedium for the organotypic tissue, and the bottom electrode(s) is/arelocated in the bottom chamber and the top electrode(s) is/are located inthe top chamber, and wherein the organotypic tissue is located betweenthe bottom electrode(s) and the top electrode(s). The present inventionalso refers to the use of the device according to the present inventionfor measuring impedance in organotypic tissue.

Furthermore, the present invention relates to a method for analyzing theeffect of test compounds on pathological and non-pathologicalorganotypic tissue by measuring the impedance of the organotypic tissue,wherein the organotypic tissue is cultured in a culture medium duringthe time of the analysis and the impedance of the organotypic tissue ismeasured at least once before and at least once after treating theorganotypic tissue with the test compound or the impedance of theorganotypic tissue treated with the test compound is compared to anon-treated organotypic tissue, wherein the impedance is measured usingat least one electrode at each of two opposing sides of the organotypictissue, and the electrodes are contacted with the culture medium or thetissue during measuring the impedance.

BACKGROUND OF THE INVENTION

A prime example for neurological degenerations is represented byAlzheimer's disease (AD). AD is a devastating dementia affectingapproximately 4 million people in Europe. It progressively destroys aperson's memory, ability to learn, to reason, make judgements,communicate, and carry out daily activities. Up to date, there is noeffective treatment or cure for Alzheimer's disease. On average, aperson dies 8 years after the first symptoms arise. Patients arecommonly treated with acetylcholinesterase inhibitors and/orNMDA-receptor antagonists. These drugs show modest clinical benefit inmild to moderate cases of Alzheimer. They are only efficient for up to12 months, as their beneficial effects fade. Nevertheless, the marketvalue for these drugs amounted to $ 4 billion in 2005 and is expected torise to approximately $ 6 billions by 2010. More importantly, theNational Institute on Aging estimated that medical care costs for the4.5 million Alzheimer patients in USA amounted to $ 100 billionannually, rising to $ 160 billions by 2010. As the number of Alzheimerpatients is expected to quadruplicate within the next 40 years, theincrease in care costs may exceed the ability of health systems toabsorb these costs. Therefore, the efficient and fast development ofAlzheimer drugs is not only eagerly anticipated by patients and thepharmaceutical industry, but it is a necessity for all industrializedcountries. The Alzheimer association estimated that a treatment thatwould efficiently delay the onset of AD for 5 years could save $ 50billion annually in the US.

Given the complex pathological mechanisms, drug development programs forneurodegenerative diseases such as Alzheimer's disease (AD), Parkinson'sdisease, Multiple Sclerosis (MS), bovine spongiform encephalopathy(BSE), Creutzfeldt Jacob disease (CJD), and various retinaldegenerations predominantly depend mainly on whole animal models whichare very expensive, laborious, and time consuming. Analyses of drugeffects or pathological mechanisms are predominately performed bycell-destructive procedures like immunocytochemical, molecularbiological, and/or proteinchemical methods.

But also non-destructive and labelling free measuring principles likeimpedance spectroscopy are known. Changes in impedance can be caused byalterations of intracellular or extracellular processes that have beeninduced by e.g. transformation of non-pathological into pathologicalform. Although a large number of transgenic animal models, ex vivocultures, or cell lines for neurodegenerative diseases have beenestablished, up to now, impedance spectroscopy has not been used for theanalysis of organotypic tissues e.g. tissues related to neuronaldegenerations. In most instances impedance-based screening has beencarried out on monolayer cultures, which have the disadvantage that theydo not take into account the three-dimensional geometry of the in vivosituation.

Since impedance spectroscopy is a non-invasive method, long-termmeasurements can be realized without influencing cellular behaviour.Hence, the cellular read out reflects real time conditions withoutdisturbing effects due to complex and long lasting physical procedures.The latter methods are well suited to study a broad range of biologicaland medical problems, however, in many cases the real cellularinformation dropped away since e.g. staining artefacts make it difficultor completely impossible to interpret the extracted cellular data. Inprinciple, tracing of biological processes in living cells can beperformed with modern labelling techniques, but hold the risk to falsifydata due to the positioning of foreign substance within the cell itself.

Impedance spectroscopy—also known as cellular dielectric spectroscopy(CDS) or electric impedance spectroscopy (EIS)—can be used to measurefrequency dependent alterations of passive electrical properties ofsingle cells by applying defined alternate currents and/or voltages.

The bio-impedance of single cells can be measured with a workingelectrode and a counter electrode. Different cellular parameters such asthe capacitance and resistance of the cell membranes as well asintracellular membranes of organelles, the resistance of theextracellular medium and intrinsic cytoplasm, the extracellular matrixand the contact between cell and electrode contribute to the overallcellular impedance. To analyze alterations of impedance of living cells,an alternate voltage is applied to a biological sample. Depending on thedielectric properties of sub-cellular compartments and molecules theapplied current can flow from an active working electrode through thecells whereby the remaining current is collected by a counter electrode.Depending on the frequency of the applied voltage, alterations ofcertain cellular compartments can be identified.

In this context it is possible to discriminate cellular behaviouraccording to their dispersions which can be divided in α-, β-, andγ-dispersion. The α-dispersion ranges from 1 Hz to 1 kHz and resultsfrom counter ions, glycocalyx, and from ion channels, whereas theβ-dispersion (1 kHz-100 MHz) is due to the cytoplasm membrane,intracellular membranes (organelles), cytosol and proteins.Additionally, the γ-dispersion (100 MHz-100 GHz) is defined by thedielectric properties of free and bound water, relaxation of chargedsubgroups, and partially by protein-protein interactions. In particular,the frequency dependent measurement of manifold cellular alterations ofboth electrical and non-electrical active cells under non-destructivereal-time conditions point out the infinite possibilities of thistechnique.

A commonly used impedance recording method is the so-called electriccell-substrate impedance sensing (ECIS) introduced by Giaever and Keese(Giaever, I., and Keese, C. R. Monitoring fibroblast behavior in tissueculture with an applied electric field. Proc Natl Acad Sci USA; 1984;81(12):3761-3764; U.S. Pat. No. 5,178,096). For ECIS, cells have to growon a small gold electrode implemented on the bottom of a culture dish.If an alternate voltage is applied between a small working electrode,attached cells, and a large counter electrode, an increased impedancecan be observed at a given time and single frequencies. ECIS has beenfurther optimized for automated, non-invasive, real-time, and highthroughput analysis (WO 2007/015878; WO 2006/104839; WO 2005/077104;WO2004/010103; Wegener et al. Impedance analysis of epithelial andendothelial cell monolayers cultured on gold surfaces. J Biochem BiophysMethods. 1996; 32(3):151-170; Ciambrone et al. S. Cellular dielectricspectroscopy: a powerful new approach to label-free cellular analysis. JBiomol Screen. 2004; 9(6):467-480). These impedance-based multi-welldevices have been used for recording of healthy, non-pathologicaladherent cells (monolayer cultures) for detecting cell attachment,detachment, migration, cell-substrate interaction, blood-brain-barrierfunction, chemotaxis, toxicology, proliferation,ligand-receptor-interaction, and apoptosis after application of testsubstances. In each of these cases cells were cultured as monolayer andanalyzed by impedance spectroscopy. However, measuring the impedance inmonolayer cultures does not provide data referring to thethree-dimensional structure of living tissue.

There are also several approaches using impedance-based sensors for usein living systems. Heroux and Bourdages have published an articleentitled “Monitoring living tissues by electrical impedancespectroscopy” (Ann Biomed Eng. 1994 May-June; 22(3):328-37). The articlerefers to the development of an electrical impedance spectroscopy (EIS)probe for monitoring cellular changes in living animals. This probecomprises two slender (0.17 mm) electrodes connected to two miniaturecoaxial cables and fixed at a distance of 5 mm from each other using aninsulating plate. For impedimetric analysis the probe was directlyimplanted in living animals for monitoring different tissues (braincortex, liver, kidney, spleen, and muscle). Impedance recording wasperformed after pentobarbital-induced respiratory and cardiac arrest.However, the above described assays using living animals are notsuitable for long term impedance measurements and do not provide anautomated screening method for drug candidates for the treatment ofspecific diseases.

WO 2006/047299 discloses an organotypic slice assay that can be used tostudy neurodegenerative diseases. The invention includes the generationof brain slice cultures and their possible analysis by HPLC, ELISA,MALDI, SELEX, gene arrays, or immunochemical assays. The invention alsodescribes electrophysiological recordings of action potentials by meansof whole-cell voltage- and current clamp technique. However, WO2006/047299 does not disclose a method or device for measuring impedancein organotypic tissue.

Since impedance-based recordings of organotypic cultures have not beenpossible so far, a device and method for simple, fast, cost effective,non-destructive, and labelling-free measurement of cellular parametersof pathological and non-pathological tissues especially obtained fromdifferent parts of the central nervous system of individual (animal orhuman being) is desired.

The methods and devices for measuring impedance in organotypic tissue asdefined in the claims overcome at least some of the problems of theprior art.

SUMMARY OF THE INVENTION

The present invention relates to a device 38 for measuring impedance inorganotypic tissue 35 comprising at least one recording chamber 29 witha liquid permeable membrane 28 supporting the organotypic tissue 35, atleast one bottom electrode 26 and at least one top electrode 34, whereinthe liquid permeable membrane 28 separates the recording chamber 29 intoa top chamber 30 and a bottom chamber 31, wherein the bottomelectrode(s) 26 is/are located in the bottom chamber 31 and the topelectrode(s) 34 is/are located in the top chamber 30, and wherein theorganotypic tissue 35 is located between the bottom electrode(s) 26 andthe top electrode(s) 34. Preferably, the device 38 comprises at least 2,further preferred at least 10, further preferred at least 50, evenfurther preferred at least 100 and most preferred at least 200 recordingchambers 29. Preferably, the bottom chamber 31 is adapted to contain orreceive liquid culture medium. Preferably, the liquid permeable membrane28 comprises an opening 33 for handling liquid so that the culturemedium 32 (for culturing the organotypic tissue 35) can be filled intothe bottom chamber 31.

In a preferred embodiment of the device 38, the top electrode(s) 34 inthe top chamber 30 is/are movable in at least two directions so it/theycan be contacted with or removed from the organotypic tissue 35 or theculture medium 32.

In an especially preferred embodiment of the device 38, the electrodes26 and 34 are interconnected by at least one multiplexer 3 and animpedance/gain-phase analyzer system 2.

In another preferred embodiment of the device 38, the liquid permeablemembrane 28 extends through all the recording chambers 29.

In an especially preferred embodiment of the device 38, the bottomelectrode(s) 26 are supported on a substrate 25 at the bottom of therecording chamber 29.

In another preferred embodiment of the device 38, the electrodes 26 and34 are individually addressable.

In another preferred embodiment of the device 38, the recording chambers29 are connected to an automated liquid handling system.

In another preferred embodiment of the device 38, the liquid handlingsystem can provide a humidified atmosphere in the recording chamber 29or the liquid handling system is placed in an CO₂ incubator.

In an especially preferred embodiment of invention, the device 38comprises a bottomless multiwell frame 9 with 1-1000 wells, wherein eachwell defines one recording chamber 29.

In another preferred embodiment, the device 38 comprises a lid 36 whichcontains an implemented multiplexer board 8.

In another especially preferred embodiment of the device 38, the bottomelectrodes 26 are connected to connection pads 22 via conductors,wherein the conductors are isolated from each other by a passivationlayer comprising silicon nitrite, silicon oxide, polyimide, or viscosepolymers.

In another preferred embodiment of the device 38, the number of bottomelectrodes 26 in the recording chamber is 1 to 256, further preferred 2to 256, further preferred 4 to 256, further preferred 20 to 256 and mostpreferred 56 to 256.

The present invention also relates to the use of the above defineddevice 38 for measuring impedance in organotypic tissue 35.

In an preferred embodiment of the invention, the impedance of theorganotypic tissue 35 is detected before, during and aftertransformation of non-pathological organotypic tissue 35 intopathological tissue 35.

The present invention also relates to a method for analyzingpathological mechanisms by measuring the impedance in organotypic tissueduring the transformation of non-pathological organotypic tissue intopathological tissue, wherein the transformation of the non-pathologicalorganotypic tissue into pathological tissue is preferably carried out byintroducing mutant genes by means of bacterial or viral vectors, knockout of genes related to specific diseases, or treatment with chemicalagents. The genes encoding proteins are preferably relevant for aneurodegenerative disease, preferably AD. The organotypic tissues can beobtained from prenatal (embryonic), postnatal and adult animals.

In another especially preferred embodiment of the invention, theorganotypic tissue represents a slice culture or explant culture derivedfrom any mammal, vertebrate and invertebrate species of embryonic,neonatal, postnatal, and adult individuals. Further preferred,organotypic neuronal slice cultures (derived from any part of the brainor retina) are used as organotypic tissues.

In another preferred embodiment, the pathological organotypic culturesare obtained from prenatal (embryonic), postnatal and adult animals bytransforming the non-pathological cultures in pathological cultures byknock out of genes related to any neurodegenerative disease, preferablyAD, by RNA interference techniques.

The present invention further relates to a method for analyzing theeffect of test compounds on organotypic tissue by measuring theimpedance of the organotypic tissue, comprising (i) culturing theorganotypic tissue in a culture medium during the time of the analysis;(ii) contacting the organotypic tissue with the test compound; (iii)optionally measuring the impedance of the organotypic tissue prior tostep (iii); (iv) measuring the impedance of the organotypic tissue atleast once after step (iii), wherein

-   -   the impedance is measured using at least one electrode at each        of two opposing sides of the organotypic tissue, and    -   the electrodes are contacted with the culture medium or the        tissue during measuring the impedance.

In a preferred embodiment of the method, the time span between the firstimpedance measurement before treating the organotypic tissue with thetest compound and the last measurement of the organotypic tissue treatedwith the test compound is at least 1 week, further preferred at least 2weeks and even further preferred at least 4 weeks.

In another preferred embodiment of the method, the impedancemeasurements of the organotypic tissue are continuously performed.

In an especially preferred embodiment of the method, the organotypictissue represents a slice culture or explant culture derived from anymammal, vertebrate and invertebrate species of embryonic, neonatal,postnatal, and adult individuals.

In another preferred embodiment of the method, the pathologicalorganotypic tissue is obtained by transformation of non-pathologicalorganotypic tissue into pathological tissue. Preferably, thetransformation of non-pathological organotypic tissue 35 intopathological tissue 35 is carried out by introducing mutant genes bymeans of bacterial or viral vectors, knock out of genes related tospecific diseases, or treatment with chemical agents.

In one preferred embodiment of the method, the pathological organotypictissues are generated by using stem cells carrying relevant mutations ormutations relevant for the onset and progression of anyneurodegenerative disease, preferably AD.

In another preferred embodiment of the method, the impedance of theorganotypic tissue 35 is measured before, during and aftertransformation of the non-pathological organotypic tissue 35 intopathological tissue 35 and before, during and after treating theorganotypic tissue with the test compound.

In another preferred embodiment of the method, the organotypic tissueused for measuring impedance is non-pathological tissue and is treatedwith test compounds to test the toxicity of the test compounds.

In an especially preferred embodiment of the method, measuring impedanceis performed by transient indirect electrode contact impedance recording(TIECIR), permanent indirect electrode contact impedance recording(PIECIR) or transient direct electrode contact impedance recording(TDECIR).

In another preferred embodiment of the method, the organotypic tissue isobtained from transgenic animals carrying mutation inducing propertiesof neurogenerative diseases selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Huntigton's disease,amyothrophic lateral sclerosis, prion diseases, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, multiple system atrophy, mild-cognitive impairment,ischemic stroke, multiple sclerosis, motor neuron diseases, nerve injuryand repair, age related macular degenerations, rod-cone dystrophy,cone-rod dystrophy, retinitis pigmentosa, glaucoma, and other retinaassociated degenerations. Preferably, the pathological organotypiccultures are obtained directly from transgenic animals carrying relevantmutations or mutation relevant for onset and progression of anyneurodegenerative disease, preferably Alzheimer's diseases, Parkinson'sdisease, Huntigton's disease, amyothrophic lateral sclerosis, priondiseases, Pick's disease, fronto-temporal dementia, progressive nuclearpalsy, corticobasal degeneration, multiple system atrophy,mild-cognitive impairment, ischemic stroke, multiple sclerosis, motorneuron diseases, nerve injury and repair, age related maculardegenerations, rod-cone dystrophy, cone-rod dystrophy, retinitispigmentosa, glaucoma, and other retina associated degenerations.

In another preferred embodiment of the method, the measuring of theimpedance is carried out by recording of frequency dependent impedancemagnitudes and phase angles before and after application of testcompounds at multiple frequencies (1 Hz-100 MHz).

In another preferred embodiment of the method, the impedance is measuredby using a device according to the present invention.

The device and method according to the present invention is suitable formeasuring impedance in pathological or non-pathological organotypictissue, wherein the three-dimensional structure of the tissue providesvaluable information with respect to pathological mechanisms in theliving beings or the effect of test compounds on living beings. Thepresent invention provides a simple, fast, cost effective,non-destructive, and labelling-free measurement of cellular parametersof pathological and non-pathological tissues especially obtained fromdifferent parts of the brain. The use of tissue instead of onlymonolayers of cells allows to obtain data which reflect thethree-dimensional structure of the tissue.

The device according to the present invention advantageously allows touse standard multiwell formats, which helps to decrease the costs forthe manufacturing of the device. Furthermore, automated liquid handlingsystems and incubators can be used.

Furthermore, the device and method according to the present invention issuitable for screening drug candidates or toxic compounds. The deviceand method can furthermore be used to analyze the effect of drugcandidates on pathological and non-pathological organotypic tissue. Thetreatment of non-pathological tissue with the drug candidates allows todetermine unwanted side effects and thereby effectively improves thedrug development and drug safety process.

The method and device according to the present invention allow thedetection of cellular alterations by measuring impedance in organotypictissue. The obtained impedance data are e.g. impedance magnitude andimpedance phase which in turn can be used to calculate the real part ofthe complex impedance. Alterations of these parameters may provideinformation about the cellular status before and after application of adrug or a toxic compound or between pathological and non-pathologicalcultures.

DETAILED DESCRIPTION OF THE INVENTION Device for Measuring Impedance

The device for measuring impedance in organotypic tissue comprises atleast one recording chamber. Preferably, the device comprises at least2, more preferably at least 6, more preferably at least 48 and mostpreferably at least 86 recording chambers. In the following, theexpression “recording chambers” is used, wherein it is understood thatdevices with only one recording chamber are also included in the presentinvention. The recording chambers contain the organotypic tissue(s)which is/are subjected to the impedance measurement. The recordingchambers furthermore contain a liquid permeable membrane, whichspatially divides the recording chambers into a top chamber and a bottomchamber. The liquid permeable membrane mechanically supports thepositioning and growth of the organotypic tissues. At least one bottomelectrode is contained in the bottom chamber of the recording chamberand at least one top electrode is contained in the top chamber of therecording chamber. The organotypic tissue can be cultured in therecording chamber, which may contain a culture medium at least in thebottom chamber, and, depending on the method for measuring impedance(described in detail below) the culture medium can also be contained inthe top chamber.

Bottom Electrodes

During measuring impedance, the bottom electrode(s) is/are in directcontact with the culture medium or possess an additional layer which isin direct contact with the culture medium. The additional layer can be apolymer coating. Preferably, the bottom electrode(s) are in directcontact with the culture medium. In the following, we refer to theexpression “bottom electrodes”, wherein it is understood that also arecording chamber with only one bottom electrode can be used. The bottomelectrodes are preferably connected to connection pads using conductorsand the connection pads are preferably connected to a multiplexer.

The bottom electrodes can be made of any material having the necessaryelectric conductivity. Suitable materials for electrode based impedancemeasurements are known in the art. Preferably, the bottom electrode ismade of gold, platinum, indium tin oxide ITO, silver, copper, iridium oralloys thereof. Depending on the used material, the thickness of thebottom electrodes is preferably between 10 nm and 1000 μm, furtherpreferred between 50 nm and 100 μm, and most preferred between 100 nmand 10 μm.

The bottom electrodes are attached to the bottom of the recordingchambers, wherein the recording chambers are preferably defined by amultiwell plate or multiwell frame. The bottom electrodes can bedeposited on the bottom of the recording chambers by means ofsemiconductor technology. Electrodes can be sputtered onto siliconoxide, polyethylen (PE), glass, or comparable substrates. FIG. 8B showsthe use of a multiwell frame which is placed on a substrate which formsthe bottom of the multiwell frame. The multiwell frame can be glued,screwed, soldered, melted, clipped, or fitted to the substrate in orderto provide a sealing which is liquid tight.

Preferably, universal microelectrode arrays are used. Those arraysconsist of 384 single substrate-integrated microelectrodes arranged in a24×16 format on the bottom plate of the device. Based on the arrangementof electrodes, the bottom plate represents an all-purpose device for allknown multiwell formats. This means, the number of electrodes per wellincreases with the size of the well. For instance, a single well of a6-well plate consists of 64 individual addressable electrodes, whereas asingle well of a 384 well comprises only one electrode at the bottom.However, if necessary the electrode size can be adapted to theappropriate well size. In this case, a single well of an 6-well platecan consist of only one large electrode and not of 64 individualelectrodes.

The bottom electrodes and conductors are electrically isolated from eachother by a passivation layer 27. This passivation may consists ofsilicon nitrite, silicon oxide, polyimide, or viscose polymers such asSU-8 (Allresist GmbH, 15344 Strausberg, Germany). The thickness of thepassivation depends on the used material and preferably ranges from 100nm to a 20 millimeters. Preferably, the bottom area of the recordingchamber covered by one or more electrodes ranges from 20-100%. Theelectrodes can have any possible geometrical form such as round,squared, ring-like, etc.

Top Electrodes

The top electrode(s) is/are located in the top chamber(s) of therecording chamber(s). In the following we refer to the expression “topelectrodes”, wherein it is understood that also recording chambers withonly one top electrode can be used. The top electrodes can be made ofthe same materials as the bottom electrodes and preferably consist ofgold or platinum. During measuring the impedance, the top electrodes arein direct contact with the organotypic tissue or the culture medium. Thetop electrodes can be of any shape suitable for measuring impedance. Askilled person is able to choose a suitable shape for the topelectrodes. Preferably, the top electrode has a stamp-like shape or apin-like shape, further preferred a stamp-like shape. Most preferably,every recording chamber contains only one top electrode.

In addition, the top electrodes can be movable in at least twodirections so that they can be contacted with or removed from theorganotypic tissue or the culture medium. Further preferred, the topelectrodes are movable perpendicular to the liquid permeable membrane.In forward direction, the electrode can be positioned directly on thesurface of the tissue or in the case of a small pin-like or needle-likeelectrode the top electrode can directly penetrate into the tissue. Theclose contact between tissue and top electrode is sufficient for thegeneration of an appropriate impedance circuit. After impedancemeasurement, the top electrode can be removed in the reversed directionto provide the upper surface of the tissue with oxygen, carbon dioxideand nitrogen. Positioning of the top electrode can be performed manuallyor automated by means of computer-assisted stepping motor. Destructionof tissue can be prevented by sensors detecting the pressure acting onthe tissues. Alternatively, the exact position of the top electrode canbe adjusted by measuring and analyzing the impedance or conductivityduring moving the electrode towards the tissue. The changes ofconductivity and impedance provide information about the intensity ofthe contact that has been generated between top electrode and tissue.

The top electrodes can be fixed to the device by any suitable means.Preferably, the top electrodes are integrated in a lid which covers therecording chamber(s).

Ground Electrodes

The recording chamber(s) can additionally comprise ground electrodes inthe bottom chamber. In this case, the bottom electrodes can be separatedby ground electrodes to minimise parasitic interferences (increasingsignal-to-noise ratio). The ground electrodes are connected viaconductors to a ground pad. The ground electrodes are preferablystripe-shaped. Preferably, the ground electrodes are connected to amultiplexer.

Electrode Setup

The electrodes (top, bottom, ground) are preferably individually orsimultaneously, further preferred individually addressable. The top,bottom and additional ground electrodes are connected to the measuringdevices via conductors. The electrodes and conductors are preferablymade of gold, platinum, indium tin oxide, silver, copper, iridium oralloys thereof (which are suited for electrode based impedancemeasurements). The conductors can be isolated by silicon nitrite,polyimide or others materials. Preferably, the electrodes and conductorsare additionally connected to a multiplexer and an impedance/gain-phaseanalyzer system. Preferably, the electrodes have a length of 1 mm to 20mm. Preferably, the electrodes have a stamp-, pin-, needle-, orspike-like shape geometric structures. It is also preferred that thenumber of electrodes of an individual well is between 1 to 256, furtherpreferred between 10 to 256, even further preferred between 30 to 256,and most preferred between 60 to 256.

The electrodes (top, bottom) can be electrically linked via connectionpads to the impedance equipment (e.g. multiplexer, impedance/gain-phaseanalyzer). The ground electrodes are preferably linked via ground padsto the impedance equipment. For measuring impedance changes oforganotypic tissues an alternate electrical current or voltage at singleor multiple frequencies are applied at least to one pair of electrodeswhich includes a top and bottom electrode.

The height (thickness) of the electrodes is preferably between 50 nm and1000 μm, and the diameter or width of electrodes is preferably between10 μm and 10 mm. Also preferred is that the electrodes compriseinterdigital electrodes. The electrode to electrode distance in thedevice is preferably at least 100 μm. The electrode area covering thebottom surface of an individual recording chamber is preferably between5 and 90%.

The conductive elements (electrodes, conductors, connection/ground padsetc.) are preferably isolated from each other by a passivation layerpreferably comprising silicon nitrite, silicon oxide, polyimide, viscosepolymers such as SU-8, or any non-conducting materials suitable forpassivation. Methods for isolation of conductive elements are well knownin the art.

Liquid Permeable Membrane

The liquid permeable membrane is biocompatible and mechanically supportsthe positioning and the growth of the organotypic tissue which iscultured in the recording chamber. The liquid permeable membrane canconsist of any material that is compatible to cells or tissues withoutaffecting cellular physiology. Preferably, the liquid permeable membraneconsists of polyethylen, polycarbonate, aluminium oxide, nitrocellulose,mixed cellulose esters, hydrophilic PTFE (polytetrafluorethene),polyethylennaphtalate, teflon, regenerated cellulose, cellulose acetate,nylon, silicon, polyethersulfone.

The liquid permeable membrane is located in the recording chamber anddivides the recording chamber into the top chamber and the bottomchamber. Preferably, the borders of the liquid permeable membrane areconnected to the recording chamber in order to be mechanically stable.For gas and medium exchange, the liquid permeable membrane preferablycontains pores. The pore sizes preferably range between 0.02 and 200 μm,further preferred between 0.02 and 100 μm, and most preferred between0.02 and 10 μm.

Additionally, the membrane can possess at least one opening, whichallows to e.g. exchange the culture medium in the bottom chamber and toadd test compounds into the culture medium of the bottom chamber.Preferably, the opening has a diameter of 100 μm to 10 mm and issuitable for handling liquids. If the recording chamber comprises a lid,it is preferred that the lid also possesses such an opening(microchannel) in order to allow to add or remove liquids even if thelid is closed. Preferably, the handling of liquids is carried out by anautomated liquid handling system. It is also preferred that the liquidpermeable membrane is situated to each recording chamber by cell culturemembrane inserts.

The pores allow the adequate diffusion and supply of the organotypictissue with nutrients that are added into the culture medium which is atleast in contact with the bottom side of the membrane. The membrane canbe integrated between the top electrode(s) and the bottom electrode(s)at any horizontal position within the recording chamber.

In a preferred embodiment the liquid permeable membrane is suitable toinduce pathological conditions in the organotypic tissue, which is in anon-pathological condition before culturing on the liquid permeablemembrane. In order to induce pathological conditions, the liquidpermeable membrane can be structured by photoactive lithography, softlithography, laser ablation, printing, stamping, sputtering and chemicalcoupling of amino acids, peptides, proteins, enzymes, nucleic acids,carbohydrates, inorganic agents, organic agents, biological activemolecules, pesticides, bacterias, fungis, yeasts, mycoplasms, bodyfluids etc and any combination of these test compounds which can bedirectly coupled or attached to the membrane. To improve the attachmentof the ex vivo tissues the surface of the membrane can also be coated.Preferred coatings are fibronectin, collagen, laminin, polylysine,arginine, ornithine, but similar substances can be used for an improvedadherence or to induce pathological effects.

Substrate

The recording chambers can additionally comprise a substrate onto whichthe electrode(s), connection pads etc. of the bottom chamber can bedeposited. The resulting substrate with the electrodes, conductors andconnection pads deposited thereon can then be used as the bottom of therecording chambers or the multiwell array, respectively. The substratecan be made of glass, quartz glass, borsilicate glass, silicon, ceramic,polymer, polyimide, polypropylene, polystyrole, polyester, polycarbonateor any other material suited for sputtering electrodes and conductors.The thickness of the substrate depends on the material used for itspreparation. If glass is used as substrate the thickness can vary fromless than a millimetre to several millimetres. In case the substrate isa polymer, it can be used as a thin foil of a few 100 microns whereonelectrodes can be deposited. The electrodes, conductors, andconnection/ground pads are preferably integrated in the bottomsubstrate. Additionally, the substrate can comprise a thin foil on itssurface to protect the electrical setup.

Lid

The device according to the present invention can additionally furthercomprise a lid which covers each or all of the re cording chambers,preferably, one lid can cover all the recording chambers. The topelectrode or a plurality of top electrodes can be integrated in the lid,wherein the top electrodes are preferably movable as described above.Suitable lids can be fabricated by computer numerical control (CNC)based milling of biocompatible plastic or by plastic injection mouldingand subsequent integration of electrodes by Microsystems Technology.

As described above, the lid preferably comprises an opening(microchannel) which allows to exchange culture medium or to addcompounds to the culture medium in the bottom chamber, even if the lidis closed. The opening in the lid is preferably between 0.5 mm and 10mm.

Impedance/Gain-Phase Analyzer

The device according to the invention can comprise a commercial orcustom impedance analyzer system. Such devices are well known in theart. HP4284, Hewlett Packard (USA); Solartron 1260A, SolartronAnalyticals (UK); Agilent 4294A, Agilent Technologies Deutschland GmbH(Germany); IviumStat Analyser, IVIUM Technologies (The Netherlands).

Additionally, the impedance/gain-phase analyzer is connected to acomputer to evaluate the obtained data.

Multiplexer

Additionally, the device can be connected to a multiplexer, which itselfis preferably connected to an impedance/gain-phase analyzer. Themultiplexer is a device that performs multiplexing, which means that itselects one or more of many signals obtained from the electrodes of thedevice and outputs that signals in a suitable manner which have to beanalyzed by the impedance/gain-phase analyzer (FIG. 2).

It is further preferred that the multiplexer is a multiplexer board andis integrated in the lid of the device. Suitable devices include, e.g.,Multiplexer NI-SCXI-1153, NationalInstruments (USA);Electrodemultiplexing Systems, ADG731, ADG725 from Analog Devices (USA).

Incubator and Liquid Handling System

To realise a high reproducibility for high content or high throughputscreening, especially for testing compounds (drug or toxic compounds)the device may combined with an automated liquid handling system thatalso provides a humidified atmosphere (e.g. 37° C., 5% CO₂, 95% air).Alternatively, the liquid handling system can be placed directly in aCO₂-incubator. Suitable Liquid Handling and Robotic systems are, e.g.:Freedom EVO®, TECAN Trading AG (Switzerland); Biomek FX Systems,Biomek®Assay Workstation, Beckman Coulter, (Germany); Biorobot 8000,Qiagen (Germany).

Multiwell Format

Although the recording chambers and the device according to the presentinvention can be individually manufactured by skilled person to containthe above described means, the device preferably comprises commerciallyavailable multiwell plates or bottomless multiwell frames. It is evenfurther preferred to use standard footprint multiwell plate formatsaccording to the Society of biomolecular screening (SBS).

Preferably, the device according to the present invention comprises astandard bottomless multiwell frame, wherein each well defines onerecording chamber. According to this, a single 384 well plate comprises384 recording chambers, a 192 plate consists of 192 recording chambersetc. Further preferred is the use of a bottomless multiwell frame with aplurality of wells, wherein the number of wells preferably rangesbetween 1 and 1000, further preferred between 6 and 384, furtherpreferred between 86 and 384. Preferably, the device according to thepresent invention comprises a bottomless 6 to 384 standard multiwellframe. FIG. 3 discloses the use of a multiwell frame which is positionedon a substrate (preferably made of glass), which forms the bottom of themultiwell frame. The substrate and other means of the device aredescribed in detail above. Preferably, the wells of the multiwell platesor multiwell frames have a diameter or width of 2 mm-35 mm and a heightof 5 mm-30 mm.

The liquid-permeable membrane can be situated at any position in thebottomless multiwell frame. Preferably, the liquid permeable membranecovers all recording chambers of the complete bottomless multiwellframes at any position between the opposite ends of the bottom-lessmultiwell frame. For example, the liquid permeable membrane can beintegrated horizontally in between to halves of the multiwell plate.

Depending on the used multiwell format, a different number ofmicroelectrodes can be integrated in the top and bottom chamber. Eachrecording chamber contains at least one top electrode, at least onebottom electrode and can additionally contain at least one groundelectrode. Accordingly, each recording chamber comprises at least twoelectrodes (top and bottom electrodes) e.g. per well of a 384 multiwellplate (384×2 electrodes, see FIG. 4). The recording chamber can alsocontain eight microelectrodes per well of a 96 multiwell plate (96×8electrodes), or 16 microelectrodes per well of a 48 multiwell plate(48×8 electrodes), or 32 microelectrodes per well on a 24 multiwellplate (24×32 electrodes), or 64 microelectrodes per well of a 12multiwell plate (12×64 electrodes), and 128 microelectrodes per well ofa 6 multiwell plate (6×128 electrodes). Alternatively, the electrodesizes can be adapted to the appropriate well size. This means, one largetop electrode and one large bottom electrode per well of the multiwellplate (6-384 well).

The electrodes of the multiwell plate can be interconnected by at leastone multiplexer that controls either the top electrodes or the bottomelectrodes, but it is also possible to multiplex both top and bottomelectrodes at once. Furthermore, the ground electrodes are alsoconnected (via conductors and ground pad) to the multiplexer and thecomputer (shown in FIG. 2).

The liquid permeable membrane can extend through all the recordingchambers. Preferably, the liquid permeable membrane is placed on top ofthe multiwell frame which itself is placed on the substrate. Theresulting chambers between the substrate and the liquid permeablemembrane are the bottom chambers. The top chamber can be provided byusing an additional multiwell frame which is placed on top of the liquidpermeable membrane and defines the top chambers (see e.g. FIG. 4). Theimpedance measurement of organotypic tissues is described in detailbelow.

Organotypic Tissue

The organotypic tissue which can be used for measuring impedanceaccording to the method and/or device of the present invention can benon-pathological or pathological organotypic tissue. According to theinvention, pathological organotypic tissue can either be directlyderived from individuals suffering from the concerned disease(preferably neurodegenerative diseases) or can be derived fromtransgenic animals.

Neurodegenerative diseases or disorders according to the presentinvention comprise Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, prion diseases,Pick's disease, fronto-temporal dementia, progressive nuclear palsy,corticobasal degeneration, cerebro-vascular dementia, multiple systematrophy, and mild-cognitive impairment. Further conditions involvingneurodegenerative processes are, for instance, ischemic stroke,age-related macular degeneration, narcolepsy, motor neuron diseases,nerve injury and repair, and multiple sclerosis.

Preferably, the neurodegenerative disease is selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, multiplesclerosis, amyotrophic lateral sclerosis, huntington's disease,tauopathies and prion diseases. Most preferably, the neurodegenerativedisease is Alzheimer's disease.

Additionally, non-pathological organotypic tissue can be used for themethod or device according to the invention. Such organotypic tissue canthen be transformed into a pathological form, preferably during themeasurment or the effect of test compounds (e.g. toxic effects) onnon-pathological organotypic tissue can be analyzed. The transformationof non-pathological into pathological organotypic tissue is described indetail below.

Organotypic cultures or explant cultures are preferably generated byusing healthy (non-pathological) or pathological neurodegenerative modelorganisms of Alzheimer's disease, Parkinson's disease, Huntigton'sdisease amyothrophic lateral sclerosis, prion diseases, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, multiple system atrophy, mild-cognitive impairment,ischemic stroke, multiple sclerosis, motor neuron diseases, nerve injuryand repair, age related macular degenerations, rod-cone dystrophy,cone-rod dystrophy, retinitis pigmentosa, glaucoma, and other retinaassociated degenerations, preferably AD.

In a preferred embodiment organotypic tissues can be generated and usedfor impedance measurements by stem cell-based tissue engineering.

The organotypic tissue preferably represents a slice culture or explantculture derived from any mammal (including primates and humans),vertebrate and invertebrate species of embryonic, neonatal, postnatal,and adult individuals. The mammal used as a tissue source can be awild-type mammal or can be a mammal that has been altered genetically tocontain and express an introduced gene. For example, the animal may be atransgenic animal, such as a transgenic mouse, that has been altered toexpress neural production of the β-amyloid precursor protein (Quon etal. (1991) Nature 35:598-607; Higgins et al. (1995) Proc Natl Acad SciUSA 92:4402-4406). In that embodiment, the animal will preferably bealtered to express a β-amyloid precursor protein that is derived orbased on human β-amyloid sequences. In one embodiment, the mammal usedas a tissue source is a transgenic mammal that has been alteredgenetically to express tau protein or a variant thereof.

The mammal used as a tissue source can be of any age. In one embodiment,the mammalian tissue source will be a neonatal mammal. The mammal usedas tissue source may have an age of about 1 to about 20 days, preferablyof about 3 to about 15 days, more preferably of about 5 to about 12days, still more preferably of about 7 to about 10 days, most preferablyof about 8 to about 9 days.

A variety of donor tissues can be used for preparing slice or explantcultures. Dissection of organotypic neuronal slice cultures can beperformed from any part of the brain or retina. Organotypic tissues ofthe retina can be cultured either as slice cultures or in toto, whereasbrain tissues are usually generated as slices cultures. The terms “sliceculture” or “organotypic tissue” or “ex vivo tissue” refers to sectionsof living tissue that can be cut in different orientations(anterior-posterior, dorsal-ventral, or nasal-temporal) and thickness.The term “explant” describes a living tissue or a piece of it thatretains the original thickness and cellular morphology. For example,retinal explants can be obtained from different regions, which includethe dorsal, ventral, nasal, or temporal part of the retina.

As used herein, the term “brain slice culture” means “organotypic brainslice culture” and refers to sections or explants of brain tissue whichare maintained in culture. A skilled person can readily employ knownmethods for preparing organotypic brain slice cultures. Organotypicbrain slice cultures can be derived from sections of the whole braintissue or from explants obtained from specific regions of the brain. Anyregion can be used to generate an organotypic brain slice culture.However, the preferred source of the organotypic brain slice culture isexplants obtained from specific regions of the brain, preferably thehippocampus region. Most preferably, the brain slice contains pyramidalneurons. Neuronal organotypic tissue can also be obtained from retinaexplants, wherein small retinal pieces can be used either from centralor peripheral parts of the retina.

Any mammal can be used as a tissue source for the explant that is usedto generate the organotypic tissue (preferably organotypic brain orretina slice culture) as long as the animal can serve as a tissue sourceand the organotypic slice culture can be established and maintained fora period sufficient to conduct the present methods. Such mammalsinclude, but are not limited to rats, mice, guinea pigs, monkeys andrabbits. Usually, the mammal is a non-human mammal. The method of theinvention may further comprise the step of obtaining an organotypictissue from the mammal, or providing an organotypic tissue culture. Themethod may further comprise the step of culturing or cultivating theorganotypic tissue prior to the impedance measurement.

The organotypic tissues can be cultured on biocompatible liquidpermeable membranes, which are integrated horizontally in between tohalves of the multiwell plate. In another embodiment, the organotypicculture can be pre-cultured on a common membrane insert of a culturedish, which may subsequently transferred to the recording chamber forimpedance measurement.

Methods for the generation of slice or explant cultures have beenreported in a number of previous studies (Förster et al., Hippocampalslice cultures, BioValley Monogr. Basel, Karger, 2005, 1:1-11, Eds.Poindron, Piguet, Förster; Hofmann et al., Organotypic cultures of therat retina, BioValley Monogr. Basel, Karger, 2005, 1:58-73, Eds.Poindron, Piguet, Förster Li et al., 1993, Neuroscience 52(4):799-813;Stoppini et al., 1991, J Neurosci Methods 37:173-182; Gahwiler, 1988,Trends Neurosci 11:484-490; Seil (1979) Review in Neuroscience4:105-177) and are well-suited for maintaining organotypic tissues onthe said membrane of the said recording chamber (FIGS. 1 and 5).

The preparation of organotypic tissue for use in the present inventionis described in the following. The brain and retina are isolated as fastas possible and preferably transferred into physiological dissectionmedia (e.g. MEM buffered with 10 mM Tris pH 7,2 for brain tissue andHEPES buffered F12 nutrient mix for retinal explants) supplemented withantibiotics. Suitable culture medium for impedance measurements areknown in the art. The choice of culture medium and culture conditionsdepends on the intended use, the source of tissue, and the length oftime before the section is used in the present method. Examples ofculture media include, but is not limited to 25% horse serum, 50%minimum essential media, 25% Hank's media, supplemented with antibioticand L-glutamine. Examples of culture condition include, but are notlimited to, 37° C., 5% CO2.

Cultures can be maintained for up to 8 weeks, under ideal conditions.However, organotypic brain slice cultures are preferably used after theyhave stabilized following the trauma of transfer to culture, but beforeonset of decline. In general, it is preferable to use the slice culturesfrom about 1 week to about 4 weeks after they have been generated.

Subsequently, brain and retina are cut into small pieces. Small regionsare separated from the tissue as slices or explants such that thesurface to volume ratio allows exchange between the center of the tissueand the media. A variety of procedures can be employed to section ordivide the brain tissues. For example, sectioning devices can beemployed. The size/thickness of the tissue section will be basedprimarily on the tissue source and the method used forsectioning/division. For example, preferred segments are from about 200to about 600 μm, preferably from about 300 to about 500 μm, mostpreferably from about 350 μm to about 450 μm in diameter and are madeusing a tissue chopper, razor blade, or other appropriatesectioning/microtome blade. Retinal slices can be cut into slices of100-400 μm thickness. Brain slices, small retinal pieces or slices aretransferred to the said membranes. The recording chamber of themultiwell plate is filled with culture medium up to the bottom line ofthe tissue slices or explants.

Although different culture media can be used, the preferred media forbrain slices consists of 50% minimum essential media, 25% Hank's media,and 25% horse serum whereas retinal explants can be maintained in DMEMcontaining 10% foetal calve serum. Both media are supplemented withL-glutamine and antibiotics. Depending on the type of tissue andexperimental strategy other culture media can be used. If medium isexchanged every two days e.g. brain slices can be maintained for up to 8and retinal explants for up to 6 weeks in a humidified atmosphere (95%air, 5% CO₂ and 37° C.). In each case, it is necessary to maintain thetissue within an air-liquid interface, which means that the organotypictissues are not covered by medium but are supplied with culture mediumfrom the bottom chamber.

At least one slice or explant can be cultured on the membrane. Inanother embodiment multiple slices or explants can be cultured on asingle membrane. In another preferred embodiment for impedancemeasurements co-culturing experiments can be performed by culturingslices or explants from different parts of the brain or from at leasttwo different tissue sources (e.g. retina and brain or retina, brain andpigmented epithelium). In each of these cases the organotypic tissue ortissues pieces cover a membrane area ranging from 1%-100%.

Transformation of Non-Pathological into Pathological Organotypic Tissue

Transformation of non-pathological into pathological organotypic tissuecan be achieved by: (i) introducing mutant genes by means of bacterialor viral vectors, wherein these genes may encode proteins that arerelevant for the development of the concerned diseases (relevantproteins for the development of AD are e.g. tau, APP, secretases); (ii)knock out of genes related to the concerned diseases (preferably AD) byRNA interference techniques; (iii) by any chemical agents that arecapable to induce specific pathological mechanisms of the abovedescribed diseases (preferably AD). For example pathological mechanismsof AD can be induced by tau-hyperphosphorylation using ocadaic acid.

The methods (i) and (ii) require to introduce polynucleotides into theorganotypic tissue and the cells thereof respectively. This is commonlyachieved by transfection or transduction methods known in the art. Themethods for transforming non-pathological organotypic tissue using thetau protein and APP are described in detail below.

Transfection and Transduction

As used herein, the term “transduction” is used to describe the deliveryand introduction of polynucleotide to eukaryotic cells using viralmediated delivery systems, such as, adenoviral, AAV, retroviral, orplasmid delivery gene transfer methods. These methods are known to thoseof skill in the art, with the exact compositions and execution beingapparent in light of the present disclosure.

As used herein, the term “transfection” is used to describe the deliveryand introduction of polynucleotide to a cell using non-viral mediatedmeans, these methods include, e.g. calcium phosphate- or dextransulfate-mediated transfection; electroporation; glass projectiletargeting; and the like. These methods are known to those of skill inthe art, with the exact compositions and execution being apparent inlight of the present disclosure.

Preferably the transfection or transduction is transient. This generallyrefers to transient expression of the DNA construct introduced into thecells. The expression of the tau protein or the variant thereof usuallypeaks at around day 7-8 post transfection or transduction.

Transfection or transduction is preferably performed about 3 days toabout 10 days, preferably about 4 days to about 6 days after theorganotypic tissue culture has been prepared. It is further preferredthat the impedance of the organotypic tissue is measured during thetransfection or transduction.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus towhich another polynucleotide segment may be operably inserted so as tobring about the replication or expression of the segment. The vector mayparticularly be a plasmid, a cosmid, a virus or a bacteriophage usedconventionally in genetic engineering that comprise a polynucleotideencoding e.g. tau protein or a variant thereof. Expression vectorsderived from viruses such as retroviruses, vaccinia virus,adeno-associated virus, herpes viruses, or bovine papilloma virus, maybe used for delivery of the polynucleotide or vector into the cells ofthe organotypic tissue. Methods which are well known to those skilled inthe art can be used to construct recombinant viral vectors; see, forexample, the techniques described in Sambrook et al., “MolecularCloning, A Laboratory Manual, 2^(nd) ed. 1989, CSH Press, Cold SpringHarbor, N.Y. and Ausubel et al., Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, the polynucleotides and vectors described herein can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the polynucleotide described herein can be transferred intothe host cell by well-known techniques, which vary depending on the typeof cellular host. Preferably, the vector is a viral vector, morepreferably it is a herpes simplex virus vector or a lentiviral vector.Vectors suitable for transfection of organotypic tissue cultures likebrain slice cultures are described, e.g. in Lilley & Coffin (2003) andLilley et al. (2001).

The term “recombinant” means, for example, that a polynucleotidesequence is made by an artificial combination of two otherwise separatedsegments of sequence, e.g., by chemical synthesis or by the manipulationof isolated polynucleotides by genetic engineering techniques.

The term “polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide that may be unmodified RNA or DNA or modifiedRNA or DNA. The polynucleotide may be single- or double-stranded DNA,single or double-stranded RNA. As used herein, the term “polynucleotide”also includes DNAs or RNAs. It will be appreciated that a variety ofmodifications may be made to DNA and RNA that serve many useful purposesknown to those of skill in the art. The term “polynucleotide” as it isemployed herein embraces such chemically, enzymatically or metabolicallymodified forms of polynucleotides, as well as the chemical forms of DNAand RNA characteristic of viruses and cells, including, for example,simple and complex cells.

The organotypic tissue, e.g. a brain slice is usually transfected ortransduced by contacting the tissue with the vector. Preferably, thetransfection or transduction is performed in a manner such thatpyramidal neurons are transfected or transduced. In addition, it ispreferred to minimize vector consumption. For that purpose, amicrodroplet is e.g. placed roughly onto the CA1 region of eachindividual slice. The microdroplet has a volume of from about 0.04 μl toabout 0.2 μl, preferably of from about 0.05 μl to about 0.15 μl, morepreferably of from about 0.06 μl to about 0.1 μl, even more preferablyof from about 0.07 μl to about 0.09 μl, most preferably of about 0.08μl. The microdroplet may be applied using a syringe, e.g. a 1 μlsyringe, such as a 1 μl Hamilton syringe. This embodiment is preferablyused for viral transduction of hippocampal slice cultures. It may beused, however, also for transfection or transduction of slices fromother brain regions, e.g. cortex or midbrain or slices from retinaexplants.

Transformation of Non-Pathological Organotypic Tissue Using App

In a specific embodiment, the transformation of non-pathological intopathological organotypic tissue comprises the step of contacting saidorganotypic tissue, e.g. a brain slice with β-amyloid precursor protein(β-APP) or a fragment or derivative or variant thereof. In anotherembodiment, the method further comprises the step of transfecting ortransducing said at least one organotypic tissue with a recombinantvector comprising a polynucleotide encoding β-amyloid precursor proteinor a fragment or derivative or variant thereof. The preferred fragmentis the β-amyloid peptide Aβ₁₋₄₂. The β-amyloid peptide is derived from alarger Type I membrane spanning protein, β-APP, which has severalalternatively spliced transcripts. The amino acid sequence of β-APP andAβ₁₋₄₂ are described in. Kang J. et al., 1987;: Knauer M. F et al.,1992; Homo sapiens APP (Gen-ID): NM 201414.

The term “fragment” as used herein is meant to comprise e.g. analternatively spliced, or truncated, or otherwise cleaved transcriptionproduct or translation product. The term “derivative” as used hereinrefers to a mutant, or an RNA-edited, or a chemically modified, orotherwise altered transcription product, or to a mutant, or chemicallymodified, or otherwise altered translation product. For instance, a“derivative” may be generated by processes such as alteredphosphorylation, or glycosylation, or, acetylation, or lipidation, or byaltered signal peptide cleavage or other types of maturation cleavage.These processes may occur post-translationally.

Transformation of Non-Pathological Organotypic Tissue Using Tau Protein

The tau protein is preferably human tau protein. The amino acid sequenceof wild type human tau protein is shown in SEQ ID NO:1. (Homo sapiensmicrotubule-associated protein tau (MAPT): NM 016834/NP_(—)058518). Theterm “variant” as used herein refers to any polypeptide or protein, inreference to polypeptides and proteins disclosed in the presentinvention, in which one or more amino acids are added and/or substitutedand/or deleted and/or inserted at the N-terminus, and/or the C-terminus,and/or within the native amino acid sequences of the native polypeptidesor proteins of the present invention. Furthermore, the term “variant”includes any shorter or longer version of a polypeptide or protein.Variants comprise proteins and polypeptides which can be isolated fromnature or be produced by recombinant and/or synthetic means. Nativeproteins or polypeptides refer to naturally-occurring truncated orsecreted forms, naturally occurring variant forms (e.g. splice-variants)and naturally occurring allelic variants. The terms “variant” and“isoform” are used interchangeably herein. In one embodiment the tauprotein or variant thereof according to the present application iscapable of causing degeneration of dendrites and/or axons upontransduction of organotypic tissue e.g. brain slices with a vectorcomprising DNA encoding said tau protein.

Various isoforms of tau protein have been described. Known tau isoformsare summarized in Mandelkow & Mandelkow (1998) or Sergeant et al. (2005)Biochimica et Biophysica Acta 1739:179-197. The amino acid sequences ofthese tau variants/mutants and the nucleotide sequences encoding themare incorporated herein by reference. Preferred tau variants inaccordance with this invention are tau mutants causing frontotemporaldementia and parkinsonism linked to chromosome 17 (FTDP-17). The mostpreferred tau variant in accordance with this invention has the mutationP301L which has been described to result in motor and behaviouraldeficits in transgenic mice, with age- and gene-dose-dependentdevelopment of NFTs. The amino acid sequence of the 0N4R isoform ofhuman tau harbouring the P301L mutation comprises the amino acidsequence as shown in SEQ ID NO:3 (amino acid sequence P301L mutant: 275VQIINKKLDLSNVQSKCGSKDNIKHVLGGGS 305). The numbering of amino acids inthe human tau sequences as used herein refers to the tau isoform having441 amino acids which is shown in SEQ ID NO:2.

cDNA sequences encoding tau proteins are known in the art (e.g. Gen-IDNM016834). The skilled person can therefore easily manipulate the DNA byknown techniques to provide polynucleotides that encode the desired tauprotein or variant thereof.

Treatment of Organotypic Tissue with Test Compounds

The method and device according to the present invention can be used formeasuring intra- and extracellular physiological parameters and theiralterations due to application of test compounds to the organotypictissue explants. The impedance of the organotypic tissue can be analyzedduring the treatment of the organotypic tissue with the test compoundsor the impedance of the treated organotypic tissue can be compared withuntreated organotypic tissues.

As used herein, the term “test compound” refers to any compound that isused for treating the organotypic tissue. The test compound can be adrug candidate and the effect of the drug candidate on pathologicalorganotypic tissue (for e.g. determining the pharmaceutical activity ofthe test compound) or non-pathological organotypic tissue (for e.g.determining side effects of the test compound) is analyzed. Furthermore,the test compound can be a potential toxic compound and the effect ofthe test compound on non-pathological tissue is analyzed.

Test compounds (especially drug candidates) that are assayed in theabove method can be randomly selected or rationally selected ordesigned. As used herein, a test compound is said to be randomlyselected when the test compound is chosen randomly without consideringthe structure of other identified active test compounds. An example ofrandomly selected test compounds is the use a chemical library, apeptide combinatorial library, a growth broth of an organism, or a plantextract.

As used herein, a test compound (especially a drug candidate) is said tobe rationally selected or designed when the test compound is chosen on anonrandom basis. Rational selection can be based on the target of actionor the structure of previously identified active test compounds.Specifically, test compounds can be rationally selected or rationallydesigned by utilizing the structure of test compounds that are presentlybeing investigate for use in treating Alzheimer's disease.

The test compounds (especially drug candidates) are preferably:peptides, small molecules, and vitamin derivatives, as well ascarbohydrates. The test compounds may be amino acids, viruses, nucleicacids, enzymes, natural or synthetic peptides or protein complexes, orfusion proteins, carbohydrates, biological active molecules, etc. Theymay also be antibodies, organic or inorganic molecules or compositions,drugs and any combinations of any of said agents above. They may be usedfor testing, for diagnostic or for therapeutic purposes. A skilledartisan can readily recognize that there is no limit as to thestructural nature of the test compounds to be used in accordance withthe present invention.

The potential toxic test compounds are preferably amino acids, peptides,proteins, enzymes, nucleic acids, carbohydrates, inorganic agents,organic agents, biological active molecules, quantum dots,nano-particles, pesticides, bacterias, fungis, yeasts, mycoplasms, bodyfluids etc. and any combination of these test compounds. Potential toxicsubstance are e.g. inorganic agents, organic agents, pesticides,bacterias, fungis, yeasts, mycoplasms, amino acids, peptides, proteins,enzymes, nucleic acids, carbohydrates, biological active molecules,quantum dots, nano-particles, body fluids etc and any combination ofthese test compounds.

At the commencement of an experiment, an organotypic tissue is typicallytransferred onto the liquid permeable membrane of the recording chamberof the device. The culture medium can either be present before theorganotypic tissue is transferred to the recording chamber or the mediumcan be added after the organotypic tissue has been transferred to therecording chamber. Preferably, the transformation of the organotypictissue or the treatment of the organotypic tissue with a test compoundis carried out at culture day 3 or later, when the tissues are stronglyattached to the liquid permeable membrane and the trauma of preparationhas healed.

The transformation and treatment of the organotypic tissue can becarried out by adding the necessary substances to the media or thetissue. In general, a test compound or any other necessary substanceswill be first dissolved in appropriate vehicle, such as, but not limitedto, DMSO, water, physiological saline, or media, to make a stocksolution and then diluted into the media. A vehicle control test may beincluded when the present invention is used.

Preferably, a range of doses of test compounds (e.g. drug candidate orpotential toxic compound) is tested. The range tested initially may beinformed by prior knowledge of the effects of the test compound orclosely related substances on purified proteins, cells in culture, ortoxicity in other test systems. In the absence of such knowledge, thedose range is preferably from about 1 nM to about 100 μM. A skilledartisan can readily develop a testing range for any particular testcompound or series of test compounds.

The test compound (e.g. drug candidate or potential toxic compound) istypically applied to the pathological organotypic tissue (obtained fromindividuals suffering from the concerned disease or transgenic animalsor derived by transformation of non-pathological into pathologicalorganotypic tissue) or to non-pathological tissue for about 4 hours toabout 21 days, preferably from about 1 day to about 7 days. In the caseof long term application, fresh medium containing the test compound canbe applied periodically; more frequently if rapid loss of test compounddue to chemical conversion or to metabolism is suspected.

Application of test compounds and medium exchange can be carried outmanually or by an automated liquid handling system. For this purpose thelid of the multiwell plate can be transiently removed or liquid can beapplied or exchanged through small microchannels integrated in the lidand membrane. The latter is needed for replacing medium or adding thetest compound to the reservoir at the bottom side of the liquidpermeable membrane.

Measuring Impedance

In a first step organotypic tissues is generated as described herein.Preferably the organotypic tissue is obtained from brain slices orretinal explants, further preferred from brain slices, more preferredfrom the hippocampus. The organotypic tissue(s) is/are transferred ontothe liquid permeable membrane(s) in the recording chamber(s). Theorganotypic tissues can be pathological or non-pathological as describedabove. In the following, we will use the expression “organotypictissues”, wherein it is understood that also only one organotypic tissuecan be used for measuring impedance. Organotypic tissue cultures aremaintained on top of the biocompatible liquid permeable membranes of therecording chambers. The organotypic tissues are then cultured in therecording chambers according to well-known methods and e.g. described inexample 1. Preferably, first impedance measurements are performed atculture day 3 or later, when the tissues are strongly attached to theliquid permeable membrane and the trauma of preparation has healed.

In an optional second step, the non-pathological (healthy) organotypictissue is transferred into pathological tissue in different ways asdescribed herein. The second step is not necessary if eitherpathological tissue is directly used, or if non-pathological tissue istreated with test compounds.

In an optional third step, application of test compounds to theorganotypic tissues can be performed.

Impedance recording can be carried out according to any known method formeasuring impedance, preferably according to the following threeprocedures: For transient indirect electrode contact impedance recording(TIECIR) the organotypic tissues are cultured with a small volume ofmedium (bottom side of the organotypic tissue has contact to the culturemedium). During impedance measurement the recording chamber istransiently filled with culture medium to cover the top electrode(s) andto guarantee the completion of the electric circuit. Thereafter, culturemedium is immediately removed up to the original level (FIGS. 5-A to5-C). The procedure can be performed several times.

For permanent indirect electrode contact recording (PIECIR) theprocedure is similar to TIECIR with the exception that the medium is notremoved and remains within the recording chamber for at least 3 days(FIGS. 5-D to 5-F).

For transient direct electrode contact recording (TDECIR) the topelectrode is movable and can be directly placed onto the organotypictissue during impedance measurement (FIGS. 5-G to 5-I). After recordingthe electrode can be traced back and re-positioned for subsequent andcontinuous recording.

The TIECIR and TDECIR are the preferred methods since they ensure airexchange on the upper surface of the biological sample which in turn isessential for maintaining viability and morphology of the organotypictissues.

During the above described steps, monitoring of impedance changes atmultiple frequencies can be carried out for preferably up to 8 weeks. Inthis way, alteration in intracellular compartments, extracellularcompartments, cell membranes, proliferation, apoptosis, differentiation,migration, phosphorylation, dephosphorylation, formation and dissolvingof tangle and plaque can be monitored. All experiments may be performedat least in triplicate to carry out appropriate statistical analysis. Tocalculate the E50 value of the test substance various concentrations canbe applied to the organotypic tissues.

Cellular parameters of the organotypic tissue (pathological ornon-pathological, optionally treated with test compounds) are detectedby recording of frequency dependent impedance magnitudes and phaseangles. Subsequent and continuous impedance recordings at a singlefrequency or multiple frequencies provide valuable information aboutcellular properties. For measuring impedance changes in organotypictissues, an alternate electrical current or voltage (1 mV-100 mV) at afrequency range of 1 Hz to 100 MHz are applied to the said electrodes.Cellular parameters can be detected by recording the frequency-dependentchanges in resistance and reactance of organotypic tissues locatedbetween at least one pair of electrodes (top and bottom electrodes).Impedance changes can be analyzed by measuring the resistant, reactance,capacitive reactance, inductive reactance and any value that can becalculated by using a combination of these parameters.

Changes in impedance can be caused by alterations of intracellular orextracellular processes that have been induced by application of testcompounds, transformation of non-pathological into pathological formetc. or by deprivation of essential components of the culture medium andreducing atmosphere.

When recording impedance, preferably the impedance magnitude, phaseangle, and normalized impedance are plotted against the frequency andtime (e.g. x−axis=time; y−axis=impedance magnitude (or phase angle, ornormalized impedance); z−axis=frequency). The impedance measurements onpathological and non-pathological organotypic cultures can becontinuously performed.

In order to minimize time consuming controlling of all individualelectrodes, the alternate current or voltage is preferably applied toall bottom electrodes. If a multiwell format is used, preferably each ofthe 6-384 top electrodes (depending on the used multiwell format) can beindividually multiplexed for impedance recording. The time needed forthe impedance measurement can be reduced further by using a multi sinusinput signal that allows parallel impedance measurement at differentfrequencies and that can be selected for the analysis of distinctcellular parameters.

Furthermore, the bottom electrodes are separated by a stripe-shapedground electrode to minimize parasitic interferences (increasingsignal-to-noise ratio) as described above. The microelectrode-basedmulti site recording of up to 384 electrodes can be realized by 2- or4-point measurements. To enable a fast and precise data read out, thealternating current is applied simultaneously to all bottom electrodes(or subsequently), whereby the data read out is achieved by multiplexingindividually controllable top electrodes (may be integrated in the lidof the multiwell plate). The impedance recording is performed in afrequency range of 1 Hz to 100 MHz with a maximal amplitude of thealternating current of 1 to 100 mV to prevent non-linear effects and toreduce thermal interferences. Cellular parameters are detected by therecording of the frequency dependent impedance magnitude and phaseangle. Both values can be normalized to control experiments and provideinformation about intra- and extracellular modifications.

Preferably, the bottom electrodes (for reference) have a stable andfixed electrode potential whereas the top electrodes (for measuring) areindividually or simultaneously addressable via the multiplexer. In analternative approach the substrate integrated electrodes at the bottomof the recording chamber can serve as measuring electrodes by using anmultiplexer, wherein the top electrodes in the top chamber can act asreference electrodes.

Method for Analyzing Impedance During Transformation of OrganotypicTissue

Based on the non-invasive and labelling-free measuring principle ofimpedance spectroscopy continuous and repeated recordings of the sametissue sample before, during and after disease-(pathology) inducingsubstances can be performed. The preparation of organotypic tissue andthe transformation of the non-pathological form into the pathologicalform during culturing of the organotypic tissue in the recording chamberis described above.

In one embodiment of the invention, the transformation ofnon-pathological into pathological organotypic tissue can be analyzed bymeasuring the impedance of the organotypic tissue before, during andafter the transformation of the organotypic tissue, preferably by usingthe device according to the present invention.

The onset and progression of pathological mechanisms on organotypictissues can be analyzed by continuous measurement of impedance or bymeasuring impedance in time intervals (e.g. every 2 hours).

After induction of pathological processes, impedance measurements overseveral weeks may provide valuable information about cellular alterationthat are directly associated with diseases like AD. Occurringalterations in impedance amplitude and phase angle at various timepoints can be related to the initial cellular situation and therebyprovide information about cellular parameters involved in the etiologyof the diseases.

In one preferred embodiment, the basic pathological mechanisms afterinduction of neurodegenerative disease (preferably AD) relevantmechanisms on hippocampal slice cultures are analyzed. However,organotypic cultures can be generated from any other part of the brainor retina. Moreover, organotypic tissue cultures can be produced fromprenatal (embryonic), postnatal and adult animals of non-vertebrate,vertebrate, mammalian, including primate species and human.

Method for Analyzing the Effect of Test Compounds on Organotypic Tissue

The method according to the present invention can be used to analyze theeffect of test compounds on organotypic tissue. In order to screen drugcandidates, the impedance of the pathological organotypic tissue ispreferably measured before, during and after treatment of the tissuewith the test compounds. In this embodiment of the invention, thepathological organotypic tissue can be obtained by any method asdescribed herein. It is also possible to analyze the effect of a drugcandidate on non-pathological tissue.

Furthermore, the method according to the present invention can be usedto analyze the effect of potential toxic compounds on non-pathologicaltissue. In this case, non-pathological organotypic tissue is treatedwith the test compounds and the impedance of the tissue is preferablymeasured before, during and after this treatment. According to allmethods of the present invention, it is also possible to compare theimpedance of the treated organotypic tissue with another non treatedorganotypic tissue.

After isolation of pathological or non-pathological slice or explantcultures and cultivation on biocompatible liquid permeable membranes ofthe recording chambers, a positive or negative effect of the testcompounds is identified by alterations of the impedance spectra. Theimpedance of the organotypic tissue can be measured before, during andafter application of test compounds (preferably up to 8 weeks) or bycomparing treated with non-treated cultures. As described above, theimpedance can be measured continuously or at selected different timepoints for data read out. Subsequent and continuous impedance recordingsat multiple frequencies (1 Hz-100 MHz) provide valuable informationabout the efficiency and safety (side effects) of the test compounds.

Pathological organotypic tissues (pathological slice or explantcultures) for impedance measurement can be obtained directly fromtransgenetic animals carrying mutations relevant for anyneurodegenerative disease (e.g. Parkinson's disease, Huntigton's diseaseamyothrophic lateral sclerosis, prion diseases, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, multiple system atrophy, mild-cognitive impairment,ischemic stroke, multiple sclerosis, motor neuron diseases, nerve injuryand repair, age related macular degenerations, rod-cone dystrophy,cone-rod dystrophy, retinitis pigmentosa, glaucoma, and other retinaassociated degenerations), preferably AD. After isolation fromtransgenetic animals, pathological slice or explant cultures can becultured and maintained within the recording chambers.

In an alternative embodiment of the invention, general toxicity ofsubstances can be analyzed by using non-neuronal organotypic tissuesderived from any part of the individual.

By using already existing pathological animal models for organotypiccultures, identification and characterization of test compounds can bedirectly performed without previous transformation of non-pathologicalinto pathological organotypic tissues.

Preferably, test compounds include drugs, liquids, water, amino acids,peptides, proteins, enzymes, nucleic acids, carbohydrates, inorganicagents, organic agents, biological active molecules, quantum dots,nano-particles, pesticides, bacterias, fungis, yeasts, mycoplasms, bodyfluids and any combination of them. In one embodiment, food andenvironmental compounds are analyzed with respect to their effect oncellular changes by impedance measurement. The invention is preferablyused to screen substances for neurotoxicity. Any potential harmfulsubstance or substances with a strong suspicion (substances derived fromenvironmental or indoor pollution) can be monitored by applying thesesubstances to the non-pathological organotypic tissues. Toxicity can beanalyzed by measuring the impedance changes before, during and afterapplication or by comparing treated with non-treated organotypiccultures. Subsequent and continuous impedance recordings at multiplefrequencies (1 Hz-100 MHz) can provide valuable information abouttoxicity.

The preferred organotypic tissues for determining toxic effects of testcompounds may be obtained from liver, heart, spleen, gut, pancreas,kidney, skin, skeleton muscle and any other organ or tissue.

The method according to the invention is also suitable for identifyingtest compounds that counteract to different pathological mechanismsrelated to neurodegenerative diseases such as Parkinson's disease,Huntigton's disease amyothrophic lateral sclerosis, prion diseases,Pick's disease, fronto-temporal dementia, progressive nuclear palsy,corticobasal degeneration, multiple system atrophy, mild-cognitiveimpairment, ischemic stroke, multiple sclerosis, motor neuron diseases,nerve injury and repair, age related macular degenerations, rod-conedystrophy, cone-rod dystrophy, retinitis pigmentosa, glaucoma, and otherretina associated degenerations), preferably to Alzheimer disease (AD).

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a single recording chamber 29.The recording chamber 29 comprises a substrate 25, a bottom electrode26, a passivation layer 27, a (biocompatible) liquid permeable membrane28, a top chamber 30, a bottom chamber 31, a culture medium 32, anopening 33 in the liquid permeable membrane 28, a top electrode 34, anorganotypic tissue 35, a lid 36, and a microchannel 37 in the lid 36.

FIG. 2 describes a measuring setup that can be used to measure impedanceof organotypic tissues 35. FIG. 2 shows the device 38, which isconnected to a multiplexer 3, an impedance/gain-phase analyzer 2, and acomputer 1. The device 38 comprises a CO₂-incubator 4, a multiwell frame5 and biological samples (organotypic tissues 35).

FIG. 3 depicts the assembly of a multiwell plate 9 for impedancerecording of organotypic tissues 35. The electrodes and the liquidpermeable membrane are not shown. The multiwell frame 5 comprises wells(not shown) and together with the glass substrate 25 defines therecording chambers 29 (not shown). On top of the multiwell frame 5 is alid 36, which contains an implemented multiplexer board 8. The substrate25 comprises connection pads 22.

FIG. 4 illustrates a schematical description of a 384 wells multiwellframe 5 with an (biocompatible) liquid permeable membrane 28 forcultivation of organotypic tissues 35. The liquid permeable membrane 28extends over all recording chambers 29. Each recording chamber 29contains a top electrode 34, a top chamber 30, a bottom chamber 31,ground electrode 18 and bottom electrodes 26. The substrate 25 (e.g.made of glass) represents the bottom of all recording chambers 29. Thesubstrate 25 comprises a passivation layer 27, connection pads 22 (whichare connected to the bottom electrodes 26), and ground pads 11 (whichare connected to the ground electrodes 18). On top of the recordingchambers 29 is a lid-integrated multiplexer board 8.

FIGS. 5-A to 5-C describe different procedures for handling oforganotypic tissues and impedance measurements.

FIGS. 6-A to 6-I illustrate organotypic hippocampal slice cultures thathave been obtained from 8-9 day old rats and subsequently cultured on abiocompatible membrane in a 6 well recording chamber. The figures show:organotypic slice cultures exposed to repeated medium overflow for 15minutes (6-A, 6-D, 6-G) or 30 minutes (6-B, 6-E, 6-F); viability testedby diamino fluorescein diacetat (6-D, 6-E, 6-F); apoptosis tested bypropidium iodide staining (6-G, 6-H, 6-I); control cultures which werenot covered with medium (6-C, 6-F, 6-I); and cultures transientlycovered with medium (6-A to 6-H).

FIGS. 7A and 7B show retinal explants isolated from 10-day old chickenembryos that were cultured for 10 days on a membrane of a 6-wellrecording chamber (FIG. 7-A). An in vivo retina of embryonic stage 20 isshown in FIG. 7-B. Abbreviations: ONL, outer nuclear layer; OPL, outerplexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer;GCL, ganglion cell layer.

FIGS. 8A and 8B illustrate a substrate 25 for the evaluation of theoptimal size of the bottom electrodes 26 for the impedance-basedmultiwell method. Bottom electrodes 26 (made of gold) of different sizesare sputtered onto a borosilicate glass substrate 25 (FIG. 8-A). Forevaluation of the bottom electrodes 26, a 384 multiwell frame 5(defining the recording chambers 29) was glued onto the substrate 25(FIG. 8-B).

FIG. 9 depicts the impedance of the bottom electrodes 26 with respect tothe size of the electrodes 26. A 6×6 frame with dimensions of a 384multiwell plate was glued onto the glass substrate and filled with 80 μlphosphate buffered saline (PBS). The impedance was recorded in afrequency range of 100 Hz to 10 MHz.

FIG. 10 illustrates the impedance of (biocompatible) liquid permeablemembranes suited for cultivation of organotypic tissues. The impedanceamplitude was recorded in a frequency range of 100 Hz to 10 MHz. Theimpedance spectra of an aluminiumoxyd membrane from TPP Switzerland(pore size 0.02 μm) and a PICM 03050 membrane from Millipore (pore size0.4) is shown. Impedance measurement without membrane.

FIG. 11 shows the measuring of impedance caused bytau-hyperphosphorylation of hippocampal slices as described in example2.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a single recording chamber 29.The recording chamber 29 comprises a substrate 25 onto which the bottomelectrode 26 and conductors (not shown) are spotted. On top of thesubstrate 25 is a passivation layer 27. The (biocompatible) liquidpermeable membrane 28 divides the recording chamber 29 into a topchamber 30 and a bottom chamber 31. During the experiment, at least thebottom chamber 31 contains a culture medium 32. The culture medium 32 isin contact at least with parts of the organotypic tissue 35, which iscultured on the liquid permeable membrane 28. The lid 36 preventsevaporation of the medium and serves as device for fixing the flexibletop electrode 34 and conductor board (not shown). The latter may acts asbasis for the multiplexer board (not shown). The lid has at least onemicrochannel 37 of 0.5 mm to 1 cm diameter and the liquid permeablemembrane 28 has an opening 33 to fill or to exchange medium, and toapply test substances or any other necessary substances. Medium exchangeand application of test compounds can be performed manually or by anautomated liquid handling system that is integrated in a CO₂ incubator.

FIG. 2 shows a schematic representation of the invention comprising acomputer 1, an impedance/gain-phase analyzer 2, a multiplexer 3, a CO₂incubator 4, and a multiwell frame 5 containing the biological samples35. The computer 1 includes software tools for controlling theimpedance/gain-phase analyzer 2, the multiplexer 3 and for acquisitionand analyses of impedance data. The impedance analyzer 2 is connected tothe electrodes (not shown) via a multiplexer 3. The multiplexer 3 isneeded for applying an alternate currents or voltages simultaneously orindividually to the electrodes of the multiwell format. For measuringimpedance changes of organotypic tissues 35 an alternate electricalcurrent or voltage (1 mV-100 mV) at a frequency range of 1 Hz to 100 MHzare applied to the said electrodes. Cellular parameters can be detectedby recording the changes in resistance and reactance of tissues locatedbetween at least one pair of electrodes. To achieve stable andreproducible impedance recordings the tissues are preferentiallycultured and maintained in a CO₂ incubator in a humidified atmosphere of5% CO₂, 95% air and 37° C. For certain tissues or to induce pathological(e.g. ischemic) conditions culture parameters can be varied (0-50% O₂,0-50% CO₂, 33-42° C.). Within the CO₂ incubator an automated liquidhandling system can be implemented.

FIG. 3 illustrates a multiwell format comprising a lid 36 that maycontain an implemented multiplexer board 8, a multiwell frame 9 that canconsist of different formats (6-, 12-, 24-, 48-, 96-, 192-, 384-wells),a substrate 25 with integrated electrodes and conductors that can belinked via connection pads 22 placed at the periphery of 25. Theelectrodes, conductors and connection pads 22 are integrated in thesubstrate 25. The connection pads 22 allow to connect the electrodes tothe multiplexer, computer etc.

FIG. 4 illustrates a more detailed schematic representation of amultiwell device according to the invention. Herein, the tissue sample35 can be cultured and maintained on a biocompatible membrane 28 of e.g.a 6-, 12-, 24-, 48-, 96-, 192-, or 384-wells plate. The impedancechanges are measured between or among at least one top electrode 34positioned in the top chamber 30 and at least one bottom electrode 26that is implemented in the bottom chamber 31 of each recording chamber29. The stripe-shaped ground electrode 18 is localized in between thebottom electrodes 26 and is suited to minimise parasitic interferences(increasing signal-to-noise ratio). The substrate 25 also containsconductors 19, wherein means 26, 18 and 19 can be sputtered onto thesubstrate 25 (e.g. made from glass). Isolation of 26, 18, and 19 can beachieved by using an appropriate passivation layer 27. An externalconnection pad 22 and a ground pad 11 provide contact to theimpedance/gain-phase analyzer. In this case, the bottom electrode 26(for reference) has a stable and fix electrode potential whereas the topelectrodes 34 (for measuring) are individually or simultaneouslyaddressable via the lid-integrated multiplexer board 8. In analternative approach the substrate integrated electrodes 26 at thebottom of the multiwell plate can serve as measuring electrodes by usingan external multiplexer, whereas the top electrodes 34 in the topchamber 30 can act as reference electrodes.

FIGS. 5-A to 5-C show the three different ways for measuring impedanceusing the method and/or the device according to the present invention.FIG. 5-A shows the transient indirect electrode contact impedancerecording (TIECIR). For TIECIR, tissues are cultured in the presence ofa small volume of medium (bottom side of tissue has contact to themedium). During impedance measurements the recording chamber istransiently filled with medium to cover the top electrode and toguarantee the completion of the impedance circuit. Thereafter, theexcess of medium is removed up to the original medium level. Thisprocedure can be performed several times without influencing viabilityand morphology.

FIG. 5-B depicts the permanent indirect electrode contact impedancerecording (PIECIR). PIECIR is comparable with TIECIR. However, forPIECIR the medium is not removed and remains within the recordingchamber for several days. This method is well-suited to induce apathological situation since neuronal ex vivo tissues such as brainslices or retinal explants show an ischemic behaviour if they are losingcontact to the air phase.

FIG. 5-C shows the transient direct electrode contact impedancerecording (TDECIR). For TDECIR, the top electrode is movable and can bedirectly placed onto the ex vivo tissue during impedance measurement.After recording, the electrode can be traced back to provide the uppertissue surface with air. In a new cycle the measuring electrode can beplaced again onto the tissue and impedance recordings can be performedagain. The process can be repeated at least 10 times a day withoutinfluencing the quality of the tissue. The contact time betweenelectrode and tissue during impedance measurement may not exceed 60minutes. In this manner, the tissue can be monitored for 6-8 weeks.

FIG. 6 illustrates organotypic hippocampal slice cultures that have beenobtained from 8-9 day old rats and subsequently cultured on abiocompatible liquid permeable membrane. After 7 days in vitro,organotypic slice cultures were exposed to repeated medium overflow for15 minutes (6-A, 6-D, 6-G) or 30 minutes (6-B, 6-E, 6-F) to simulateconditions that can be found during TIECIR, PIECIR, and TDECIR (compareFIG. 5). The procedure was carried out over a period of four days.Thereafter, viability was tested by diamino fluorescein diacetat (6-D,6-E, 6-F), while cell death was analyzed by propidium iodide (6-G, 6-H,6-I). Comparison of control cultures (6-C, 6-F, 6-I) with cultures thatwere not covered with medium (6-A to 6-H) showed no significant changesin viability and cell death. This means, that all three proceduresneeded for TIECIR, PIECIR, and TDECIR) are well-suited for culturing andrecording of organotypic tissues.

For the generation of retinal explant cultures, we used retina of 10-dayold chicken embryos. The retina was placed either in toto or in pieceson the top of a polycarbonate membrane (3 μm pore size). The cultureperiod can be extended up to 6 weeks. The medium is exclusivelylocalized in the bottom compartment and was changed every two days.Similar to hippocampal slice cultures retinal explants showed noincrease in cell death.

FIGS. 7A and 7B show retinal explants isolated from 10 day old chickenembryos that have been cultured for 10 days on a membrane of a 6-wellrecording chamber. The retinal explants were harvested, fixed and cutinto 20 μm thick sections with a cryotome. To visualise the quality ofthe morphology and lamination of retinal layers, sections were stainedwith cytox green, which specifically stains cell nuclei (FIGS. 7A, 7B).By comparison of 20 days old organotypic cultures (FIG. 7A) with in vivoretina of embryonic stage 20 (FIG. 7B), we found no significantdifferences.

FIGS. 8A and 8B illustrate the evaluation of the optimal electrode sizefor the impedance-based multi-well assay. Borosilicate glasses were usedas substrate (FIG. 8A) for fabrication of the sensor chip.

FIG. 9 depicts the impedance of the bottom electrodes 26 with respect tothe size of the electrodes 26. To determine the electrode impedance inrespect to the electrode size, a 6×6 frame of a 384 well plate was gluedonto the glass substrate and filled with 80 μl phosphate buffered saline(PBS). The impedance was recorded in a frequency range of 100 Hz to 10MHz by applying a voltage of 10 mV across the substrate integratedelectrode and a top electrode that has been dipped from the top into thePBS. As expected the impedance magnitude decreased as the diameter ofthe electrodes increased.

FIG. 10 illustrates the impedance of membranes within the recordingchamber. The impedance was recorded in a frequency range of 100 Hz to 1MHz by applying a voltage current of 10 mV across the substrateintegrated electrode and a top electrode that has been dipped from thetop into the PBS. Two membranes of different materials and pore sizeswere tested. The impedance spectra of an aluminiumoxyd membrane from TPPSwitzerland (pore size 0.02 μm) and a PICM 03050 membrane from Millipore(pore size 0.4) is shown in FIG. 10. Both types of membranes havesimilar impedance magnitudes. Surprisingly, impedance measurements thatwere carried out only in PBS (without membranes) were slightly lowerthan those performed with membranes.

FIG. 11 shows the measuring of impedance caused bytau-hyperphosphorylation of hippocampal slices as described in example2. Six to eight hippocampal slices of 400 μm thickness were placed onaluminiumoxyd membranes (0.02 μm pore size) of a 6-well recordingchamber.

Example 1 Preparation of a Substrate with Integrated Electrodes

Borosilicate glasses were used as substrate (FIG. 8A) for fabrication ofthe sensor chip. The substrates were washed thoroughly with a lint freecotton wool tip subsequently in water, acetone, propanol and waterfollowed by incubation in a solution of 96% sulphuric acid in 30%hydrogen peroxide and two wash steps in a water bath cascade. Thesubstrate was centrifuged and dried on a hot plate. Substrate coatingwas performed by centrifugation. For this purpose 500 μl of positivephoto resist was applied in the centre of the substrate. The subsequentcentrifugation dispersed the lacquer equally over the substrate yieldinglayer of a thickness of 3 μm. The positive photo resist coat was driedfor a minimum of 5 minutes on a hot plate. By means of a chrome mask thebrim of the substrate radiated by UV-light for 10 seconds. Next thesubstrate was put into developer to remove the lacquer, followed by awashing step in a water bath cascade and centrifugation. The pattern forthe electrodes and interconnects was removed from the lacquer coat withanother chrome mask repeating the steps before and drying on a hotplate. The substrate was sputtered with a 100 nm thick layer of gold todeposit electrodes and interconnects. Next the substrate was placed inacetone and rinsed in water to remove the polymerized positive-photoresist, getting a substrate with gold electrodes and interconnects. Thesubstrate was dried with a stream of N2 and placed on a hot plate. Thepassivation was performed to isolate the interconnects and embed theelectrodes. Therefore, 1 ml of SU-8 was applied in the centre of thesubstrate and centrifuged to dispense the lacquer to 1.5 μm thick filmfollowed by drying on a hot plate. With a folia mask covering theelectrodes, the passivation-layer was radiated with UV-light for 20seconds. Then the substrate was incubated on a hot plate for 1 minutesat 60° C., 1 minutes 95° C. and put into beaker with photo-developer for1 minutes. The chip was washed in propanol to remove all traces abovethe electrodes of the negative photo resist layer. Finally, the chip wasdried under a stream of nitrogen and evaluated microscopically. Goldelectrodes electrodes have diameters of 100, 300, 600, 900, 1200, and1500 μm while interconnects have a width of 10 μm. The resultingsubstrate can be used as the bottom of the multiwell frame.

Example 2 Induction of pathological tau-hyperphosphorylation andimpedance measurement

The impedance changes caused by tau-hyperphosphorylation (FIG. 11) ofhippocampal slices that were prepared from 8-9 day old rats weremeasured using the device according to the present invention. Six toeight hippocampal slices of 400 μm thickness were places on aaluminiumoxyd membrane (0.02 μm pore size) of a 6-well recording chamberand cultured for 7 days in 50% minimum essential media, 25% Hank'smedia, and 25% horse serum supplemented with L-glutamine andantibiotics. Impedance recording was performed by using a Agilent 4294Aimpedance analyser (Agilent Technologies Deutschland GmbH, Germany) incombination with a multiplexer (NI-SCXI-1153, National Instruments,USA).

24 hours before impedance measurement was carried out, medium replacedby fresh culture medium. The impedance was recorded in a frequency rangeof 100 Hz to 1 MHz by applying a voltage of 10 mV across a large topelectrode and 60 substrate integrated bottom electrodes of 30 μm indiameter.

For measuring hippocampal slice cultures, the TIECIR method was used.Organotypic slice cultures were transiently covered with medium duringimpedance measurement, which usually takes less than 30 seconds for asingle bottom electrode. If all 60 substrate integrated electrodes wereused for recording, the complete measurement takes approximately 20 minfor a frequency range of 100 Hz to 1 MHz (with 5 measuring points perfrequency). For better illustration, the impedance spectra in FIG. 11shows only three frequencies that were recorded over 16 hours by TIECIRwith a single top and bottom electrode. The normalized impedance(normalized against non-covered membrane) of hippocampal slice cultureswas stable for 9 hours. After application of 1 μM ocadaic acid, whichinduces tau-hyperphosphorylation, impedance was significantly droppeddown to 10% and remained small within the next seven hours. For repeatedimpedance measurements by TIECIR the complete medium was exchange andfresh ocadaic acid has been applied.

1. A device for measuring impedance in organotypic tissue comprising atleast one recording chamber with a liquid permeable membrane supportingthe organotypic tissue, at least one bottom electrode and at least onetop electrode, said device characterized in that: the liquid permeablemembrane divides the recording chamber into a top chamber and a bottomchamber, wherein the bottom electrode(s) is/are located in the bottomchamber and the top electrode(s) is/are located in the top chamber, andwherein the organotypic tissue is located between the bottomelectrode(s) and the top electrode(s).
 2. The device according to claim1, characterized in that the top electrode(s) in the top chamber is/aremovable in at least two directions so it/they can be contacted with orremoved from the organotypic tissue or the culture medium.
 3. The deviceaccording to claim 1, characterized in that the electrodes areinterconnected by at least one multiplexer and an impedance/gain-phaseanalyzer system.
 4. The device according to claim 1, characterized inthat the liquid permeable membrane extends through all the recordingchambers.
 5. The device according to claim 1, characterized in that thebottom electrode(s) are supported on a substrate at the bottom of therecording chamber.
 6. The device according to claim 1, characterized inthat the electrodes are individually addressable.
 7. The deviceaccording to claim 1, characterized in that the recording chamber isconnected to an automated liquid handling system.
 8. The deviceaccording to claim 7, characterized in that the liquid handling systemcan provide a humidified atmosphere in the recording chamber or theliquid handling system is placed in an CO₂ incubator.
 9. The deviceaccording to claim 1, further comprising a bottomless multiwell framewith 1-1000 wells, wherein each well defines one recording chamber. 10.The device according to claim 1, characterized in that the devicecomprises a lid which contains an implemented multiplexer board.
 11. Thedevice according to claim 1, characterized in that the bottom electrodesare connected to connection pads via conductors, wherein the conductorsare isolated from each other by a passivation layer comprising siliconnitrite, silicon oxide, polyimide, or viscose polymers.
 12. The deviceaccording to claim 1, characterized in that the number of bottomelectrodes in the recording chamber is 4 to
 256. 13. The deviceaccording to claim 1, characterized in that the liquid permeablemembrane comprises an opening for handling liquid.
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. (canceled)
 18. A method for analyzing theeffect of test compounds on organotypic tissue by measuring theimpedance of the organotypic tissue, comprising (i) culturing theorganotypic tissue in a culture medium during the time of the analysis;(ii) contacting the organotypic tissue with the test compound; (iii)optionally measuring the impedance of the organotypic tissue prior tostep (iii); (iv) measuring the impedance of the organotypic tissue atleast once after step (iii), wherein the impedance is measured using atleast one electrode at each of two opposing sides of the organotypictissue, and the electrodes are contacted with the culture medium or thetissue during measuring the impedance.
 19. The method according to claim18, characterized in that the time span between the first impedancemeasurement prior to contacting the organotypic tissue with the testcompound and the last measurement of the organotypic tissue treated withthe test compound is at least 1 week.
 20. The method according to claim18, characterized in that the impedance measurements of the organotypictissue are performed continuously.
 21. The method according to claim 18,characterized in that the organotypic tissue represents a slice cultureor explant culture derived from any mammal, vertebrate and invertebratespecies of embryonic, neonatal, postnatal, and adult individuals. 22.The method according to claim 18, characterized in that the organotypictissue is transformed into pathological tissue by by i) introducingmutant genes by means of bacterial or viral vectors, ii) knock out genesrelated to specific disease, or iii) treatment with chemical agents. 23.The method according to claim 22, characterized in that the impedance ofthe organotypic tissue is measured prior to and after transformation ofthe non-pathological organotypic tissue into pathological tissue as wellas prior to and after contacting the organotypic tissue with the testcompound.
 24. The method according to claim 18, characterized in thatthe organotypic tissue used for measuring impedance is anon-pathological tissue and is treated with test compounds to test thetoxicity of the test compounds.
 25. The method according to claim 18,characterized in that the recording is performed by transient indirectelectrode contact impedance recording (TIECIR), permanent indirectelectrode contact impedance recording (PIECIR) or transient directelectrode contact impedance recording (TDECIR).
 26. The method accordingto claim 18, characterized in that the organotypic tissue is obtainedfrom transgenic animals carrying mutation inducing properties ofneurogenerative diseases selected from the group consisting ofAlzheimer's disease, Parkinson's disease, Huntigton's disease,amyothrophic lateral sclerosis, prion diseases, Pick's disease,fronto-temporal dementia, progressive nuclear palsy, corticobasaldegeneration, multiple system atrophy, mild-cognitive impairment,ischemic stroke, multiple sclerosis, motor neuron diseases, nerve injuryand repair, age related macular degenerations, rod-cone dystrophy,cone-rod dystrophy, retinitis pigmentosa, glaucoma, and other retinaassociated degenerations.
 27. The method according to claim 18,characterized in that the measuring of the impedance is carried out byrecording of frequency dependent impedance magnitudes and phase anglesbefore and after application of test compounds at multiple frequencies(1 Hz-100 MHz).
 28. The method according to claim 18, characterized inthat the impedance is measured by using a device comprising at least onerecording chamber with a liquid permeable membrane supporting theorganotypic tissue, at least one bottom electrode and at least one topelectrode, said device characterized in that: the liquid permeablemembrane divides the recording chamber into top and a bottom chamber,wherein the bottom electrode(s) is/are located in the bottom chamber andthe top electrode(s) is/are located in the top chamber, and wherein theorganotypic tissue is located between the bottom electrode(s) and thetop electrode(s).